We have demonstrated the presence of a gene family of mammalian Na+/H+ exchangers by cloning and sequencing three cDNAs from a mammalian small intestinal cDNA library and stably expressing the corresponding isoforms for two of these. One of these is found in all mammalian cells, is on the basolateral membrane of intestinal cells and appears to be involved in housekeeping functions. The other two appear to be epithelial isoforms, probably involved in Na+ absorption. This grant will predominantly probe structure/function relationships of these three isoforms in order to begin understanding how these proteins carry out their functions. This will be done using molecular biologic techniques plus measuring intracellular pH via fluorescent dyes. The three Na+/H+ exchangers stably expressed in the fibroblast cell line, PS120, which is devoid of endogenous Na+/H+ exchange, will be characterized as to 1) K Na+; 2) K EIPA; 3) H+ modifier site; 4) regulation by protein kinases; 5) role of glycosylation. Chimeric cDNAs and mutational analysis of the three cloned Na+/H+ exchangers will be made and stably expressed in PS120 fibroblasts in order to determine which part of the Na+/H+ exchanger: 1) defines amiloride sensitivity; 2) does the C- terminal cytoplasmic domain of the epithelial exchanger determine regulation of Na+/H+ exchange; 3)accounts for the internal H+ modifier site. In addition, advantage will be taken of our preliminary observation that an epithelial cell line, Caco-2, has only the housekeeping Na+/H+ exchanger that it sorts only to the basolateral membrane. The two epithelial Na+/H+ exchangers will be transfected separately into Caco-2 cells and it will be established whether they sort to the Caco-2 apical membrane. By comparing regulation of the epithelial Na+/H+ exchanger in the fibroblast and Caco-2 cell, what the apical membrane location or presence in an epithelial cell contributes to regulation of Na+/H+ exchange will be studied. Whether there are additional members of the Na+/H+ exchanger gene family, with an emphasis on epithelial tissues, will be studied by further screening the rabbit ileal villus epithelial cDNA library via differential hybridization.